Tissue factor pathway inhibitor (TFPI) produces factor Xa (FXa)-dependent feedback inhibition of factor VIIa/tissue factor (FVIIa/TF)-induced coagulation. In humans, TFPI is expressed as two isoforms. TFPI1 contains three tandem Kunitz-type protease inhibitor domains, Kunitz-1 binds FVIIa in the FVIIa/TF complex and the second binds FXa. Kunitz-3 does not possess protease inhibitor activity. TFPI2 lacks the Kunitz-3 and the C-terminus of TFPI1. The latter is required for TFPI1's optimal inhibition of FXa and its anticoagulant activity in one-stage coagulation assays. TFPI1 interacts with protein S (PS), which enhances its anti-FXa activity, and with factor V (FV), which may prolong its clearance from plasma. Both forms of TFPI associate with the surface of cells in a glycosylphosphatidylinositol (GPI)-anchor dependent fashion, but through different mechanisms. TFPI1, present in plasma and secreted by activated platelets, appears to bind to another GPI-anchored protein(s), whereas TFPI2 contains an intrinsic GPI-anchor. We plan to determine the structures within TFPI1 responsible for its binding to PS and FV and further explore its interactions with cell surfaces. The form(s) of TFPI, which circulate bound to lipoproteins, will be identified and the relevance of a potential additional isoform of TFPI, TFPI4, will be investigated. TFPI gene-disrupted mice (TFPI KO) die intrautero of widespread thrombosis and a consumptive coagulopathy, but are rescued by a low level of either of two transgenes with FVIIa/TF inhibitory activity. One, Tie2-hTFPI1, directs expression in endothelial cells, the other, Tf-mXK1-hAlb directs expression into plasma. Transgene-rescued TFPI KO mice and appropriate bone marrow transplantation studies will be used to assess the role of platelet- associated TFPI1 and to determine the effect of low levels of endogenous TFPI on thrombosis, inflammation, and atherosclerosis using mouse models. Public Health Relevance: TFPI is a plasma and cell-associated protein, which exists as two isoforms (TFPI1 and TFPI2) and plays an important role in regulating the initiation of coagulation. The goals of this proposal are to determine the mechanism and relevance of its interactions with circulation lipoproteins, cell surfaces, and two other coagulation-related proteins, Protein S and Factor V, and to determine the effects of TFPI deficiency on thrombosis, inflammation, and atherosclerosis using mouse models.